Leaf waxes protect terrestrial plants from biotic and abiotic stresses and are important sedimentary biomarkers for terrestrial plants. Thus, understanding the production and ablation of leaf waxes is critical in plant physiology and for geochemical studies. However, there have been no accurate approaches to quantify leaf wax production at different time scales. In this study, we demonstrate a novel approach to study leaf wax regeneration by irrigating plants with a pulse of deuterium-enriched water, followed by measurements of leaf wax D/H ratios by gas chromatography/isotope-ratio mass spectrometry (GC/IRMS). We demonstrate the efficacy of this approach using the grass species Phleum pratense in a greenhouse environment. Using a binary isotope mass balance model, we are able to quantify the regeneration rates of the C16, C18 acids and leaf waxes (C23–C31n-alkanes; C22–C30n-acids) over a diurnal cycle. Our results show that within one day 33–47 % of C16 and C18 acids are regenerated, and thus the recycling time for these compounds is 2–3 days. For C22–C26n-alkyl lipids, 7–21 % are regenerated within one day and thus they require 5–16 days to recycle. In comparison, the recycling time for long-chain n-alkyl lipids (C27–C31) is as long as 71–128 days. Our approach can be applied to different plants at shorter or longer time scales by adjusting the degree of isotopic labeling, sampling intervals and the amount of irrigation water. Copyright © 2011 John Wiley & Sons, Ltd.